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Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase

View Article: PubMed Central - PubMed

ABSTRACT

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotidereductase, leading to lowered cellular pools of deoxyribonucleosidetriphosphates. The reduced levels for DNA precursors is believed to causereplication fork stalling. Upon treatment of the hyperthermophilic archaeonSulfolobus solfataricus with HU, we observe dose-dependentcell cycle arrest, accumulation of DNA double-strand breaks, stalled replicationforks, and elevated levels of recombination structures. However,Sulfolobus has a HU-insensitive class II ribonucleotidereductase, and we reveal that HU treatment does not significantly impactcellular DNA precursor pools. Profiling of protein and transcript levels revealsmodulation of a specific subset of replication initiation and cell divisiongenes. Notably, the selective loss of the regulatory subunit of the primasecorrelates with cessation of replication initiation and stalling of replicationforks. Furthermore, we find evidence for a detoxification response induced by HUtreatment.

No MeSH data available.


ChIP Analyses of Chromosome Occupancy by Replication Initiation and DNARepair Factors Modulated by HU Treatment(A–D) ChIP analyses of binding of the Orc1-3 and WhiP proteins to thethree replication origins (A–C) and distal control locus SSO2847 (D) incells treated with 0 or 10 mM HU for 7 hr. ChIP reactions were performed intriplicate, and data are expressed as fractional recovery of the total inputmaterial.(E–H) Occupancy of the indicated genomic loci by MCM (E), Alba (F), Hel308(G), and RadA (H) as adjudged by ChIP from untreated cells or cells treated with5 or 10 mM HU for 4 hr.Mean values of the triplicate repeats are shown, and error bars indicate the SDof the data.
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Figure 4: ChIP Analyses of Chromosome Occupancy by Replication Initiation and DNARepair Factors Modulated by HU Treatment(A–D) ChIP analyses of binding of the Orc1-3 and WhiP proteins to thethree replication origins (A–C) and distal control locus SSO2847 (D) incells treated with 0 or 10 mM HU for 7 hr. ChIP reactions were performed intriplicate, and data are expressed as fractional recovery of the total inputmaterial.(E–H) Occupancy of the indicated genomic loci by MCM (E), Alba (F), Hel308(G), and RadA (H) as adjudged by ChIP from untreated cells or cells treated with5 or 10 mM HU for 4 hr.Mean values of the triplicate repeats are shown, and error bars indicate the SDof the data.

Mentions: Next, we employed chromatin immunoprecipitation (ChIP) to test whetherthe residual Orc1 initiator proteins remain associated with origins following HUtreatment. However, as can be seen in Figures4A–4C, neither Orc1-1 nor Orc1-3 binds detectably to theorigins following HU treatment. In contrast, WhiP, the levels of which are notaffected by HU treatment, remains associated with oriC3 (Figure 4C). Next, we performed ChIP analysisof the replicative helicase, MCM (Figure4E). We observe up to 25-fold enrichment of MCM at both origin proximaland origin distal loci following HU treatment. This is not simply due toimproved cross-linking or DNA recovery following HU treatment as ChIP testingthe distribution of the chromatin protein Alba shows at most a 2.5-foldvariation between treated and non-treated samples (Figure 4F). We propose therefore that the accumulation of MCMcorresponds to elevated levels of stalled replication forks following HUtreatment (Figure 3B). As discussed in theIntroduction, biochemical studies have suggested that the essential Hel308helicase may be involved in processing of stalled replication forks (Woodman and Bolt, 2009). We thereforeperformed ChIP analyses with antisera generated against this protein andobserved an enrichment of up to 22-fold at origins of replication (Figure 4G). Interestingly, this protein wasonly modestly enriched (4.6-fold) at the non-origin locus where we observed25-fold enrichment of MCM. Finally, we performed ChIP with the RecA/Rad51-likerecombinase, RadA, and observed up to 7-fold enhancement of this protein atorigins and distal loci upon HU treatment (Figure4H).


Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase
ChIP Analyses of Chromosome Occupancy by Replication Initiation and DNARepair Factors Modulated by HU Treatment(A–D) ChIP analyses of binding of the Orc1-3 and WhiP proteins to thethree replication origins (A–C) and distal control locus SSO2847 (D) incells treated with 0 or 10 mM HU for 7 hr. ChIP reactions were performed intriplicate, and data are expressed as fractional recovery of the total inputmaterial.(E–H) Occupancy of the indicated genomic loci by MCM (E), Alba (F), Hel308(G), and RadA (H) as adjudged by ChIP from untreated cells or cells treated with5 or 10 mM HU for 4 hr.Mean values of the triplicate repeats are shown, and error bars indicate the SDof the data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134839&req=5

Figure 4: ChIP Analyses of Chromosome Occupancy by Replication Initiation and DNARepair Factors Modulated by HU Treatment(A–D) ChIP analyses of binding of the Orc1-3 and WhiP proteins to thethree replication origins (A–C) and distal control locus SSO2847 (D) incells treated with 0 or 10 mM HU for 7 hr. ChIP reactions were performed intriplicate, and data are expressed as fractional recovery of the total inputmaterial.(E–H) Occupancy of the indicated genomic loci by MCM (E), Alba (F), Hel308(G), and RadA (H) as adjudged by ChIP from untreated cells or cells treated with5 or 10 mM HU for 4 hr.Mean values of the triplicate repeats are shown, and error bars indicate the SDof the data.
Mentions: Next, we employed chromatin immunoprecipitation (ChIP) to test whetherthe residual Orc1 initiator proteins remain associated with origins following HUtreatment. However, as can be seen in Figures4A–4C, neither Orc1-1 nor Orc1-3 binds detectably to theorigins following HU treatment. In contrast, WhiP, the levels of which are notaffected by HU treatment, remains associated with oriC3 (Figure 4C). Next, we performed ChIP analysisof the replicative helicase, MCM (Figure4E). We observe up to 25-fold enrichment of MCM at both origin proximaland origin distal loci following HU treatment. This is not simply due toimproved cross-linking or DNA recovery following HU treatment as ChIP testingthe distribution of the chromatin protein Alba shows at most a 2.5-foldvariation between treated and non-treated samples (Figure 4F). We propose therefore that the accumulation of MCMcorresponds to elevated levels of stalled replication forks following HUtreatment (Figure 3B). As discussed in theIntroduction, biochemical studies have suggested that the essential Hel308helicase may be involved in processing of stalled replication forks (Woodman and Bolt, 2009). We thereforeperformed ChIP analyses with antisera generated against this protein andobserved an enrichment of up to 22-fold at origins of replication (Figure 4G). Interestingly, this protein wasonly modestly enriched (4.6-fold) at the non-origin locus where we observed25-fold enrichment of MCM. Finally, we performed ChIP with the RecA/Rad51-likerecombinase, RadA, and observed up to 7-fold enhancement of this protein atorigins and distal loci upon HU treatment (Figure4H).

View Article: PubMed Central - PubMed

ABSTRACT

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotidereductase, leading to lowered cellular pools of deoxyribonucleosidetriphosphates. The reduced levels for DNA precursors is believed to causereplication fork stalling. Upon treatment of the hyperthermophilic archaeonSulfolobus solfataricus with HU, we observe dose-dependentcell cycle arrest, accumulation of DNA double-strand breaks, stalled replicationforks, and elevated levels of recombination structures. However,Sulfolobus has a HU-insensitive class II ribonucleotidereductase, and we reveal that HU treatment does not significantly impactcellular DNA precursor pools. Profiling of protein and transcript levels revealsmodulation of a specific subset of replication initiation and cell divisiongenes. Notably, the selective loss of the regulatory subunit of the primasecorrelates with cessation of replication initiation and stalling of replicationforks. Furthermore, we find evidence for a detoxification response induced by HUtreatment.

No MeSH data available.